Developing Single-Molecule TPM Experiments for Direct Observation of Successful RecA-Mediated Strand Exchange Reaction

Developing Single-Molecule TPM Experiments for Direct Observation of Successful RecA-Mediated Strand Exchange Reaction

RecA recombinases play a central role in homologous recombination. Once assembled on single-stranded (ss) DNA, RecA nucleoprotein filaments mediate the pairing of homologous DNA sequences and strand exchange processes. We have designed two experiments based on tethered particle motion (TPM) to investigate the fates of the invading and the outgoing strands during E. coli RecA-mediated pairing an...

Direct Single-Molecule Observation of Mode and Geometry of RecA-Mediated Homology Search.

Genomic integrity, when compromised by accrued DNA lesions, is maintained through efficient repair via homologous recombination. For this process the ubiquitous recombinase A (RecA), and its homologues such as the human Rad51, are of central importance, able to align and exchange homologous sequences within single-stranded and double-stranded DNA in order to swap out defective regions. Here, we...

Improved RecA-assisted fluorescence assay for DNA strand exchange reaction.

MutS inhibits RecA-mediated strand exchange with platinated DNA substrates.

Human cell lines and Escherichia coli dam mutants are sensitive to the cytotoxic action of the anticancer agent, cisplatin. Introduction of mutations disabling DNA mismatch repair into these cell lines renders them resistant to the action of this drug. We used RecA-mediated strand exchange between homologous phiX174 molecules, one that was platinated and the other that was unmodified, to show t...

DNA Strand Exchange Promoted by RecA K72R

Replacement of lysine 72 in RecA protein with arginine produces a mutant protein that binds but does not hydrolyze ATP. The protein nevertheless promotes DNA strand exchange (Rehrauer, W. M., and Kowalczykowski, S. C. (1993) J. Biol. Chem. 268, 1292–1297). With RecA K72R protein, the formation of the hybrid DNA product of strand exchange is greatly affected by the concentration of Mg in ways th...